I need suggestion about my current problem. I purified serine hydroxymethyltransferase protein using Ni-NTA resin and enough amounts and purity of protein was observed on SDS-PAGE. But the protein was precipitated soon after purification. I used 20 mM Tris pH8.5, 500 mM NaCl, buffer for sonication of E. Coli, Ni-NTA binding and washing of resin. Then protein was eluted by 20 mM Tris pH8, 500 mM NaCl, 200 mM imidazole. And after purification immediately I was added 1mM DTT and 5mM EDTA.
Hi,
I will suggest to check the pI (isoelectric point) of the protein you are purifying. It might be that the pH of your buffer is similar to protein pI and in such case the proteins offen precipitate. Solution will be to use the buffer with pH of at least 1 unit away from pI.
Hope it will help.
Regards
Dear Wojciech Paslawski
Thanks for your suggestion. The pI of that protein is 7.3.That why i choose pH 8, 8.5,and 6.5. But in all case protein gets precipitation. If you have another idea, please share with me. Thanks
Dear Md.Rezwanul Haque,
quite often addition of a certain percentage of a stabilizing osmolyte like glycerol or sucrose (5%-30%, to be tested) helps preventing protein aggregation. Salt concentration can also play a role. A very nice compilation of tips and tricks to prevent protein aggregation can be found at https://www.biozentrum.unibas.ch/fileadmin/redaktion/Forschung/Research_Groups/BF/Protocols/Preventing_Protein_Aggregation.pdf
In your specific case you might also try to remove the imidazole after elution from the Ni-NTA (e.g. by dialysis or buffer exchange via gel filtration)...some proteins don't like this reagent at high concentrations.
Hope you'll find the right solution for your protein!
Best regards
Johann
Hi once more,
in such case I am afraid it might be many factors and you need to check them step by step.
First of all I will check if the precipitation you observe is not caused by protein degradation. Sometimes EDTA is not enough and you might need to add protease inhibitors.
You can also check if your protein is not aggregating by any chance due to high concentration. Spin down the precipitate and try to dissolve it in buffer with high concentration of Urea. If that is the case you need to keep your protein more diluted while in solution.
And it might be also that the buffer is still not optimal for your protein. This will force a bit more work including checking different salt concentrations (maybe 500 mM is too high for your protein) and going even further away from isoelectric point with pH.
Best regards,
Wojciech
I would say the pH of your buffer is close to protein PI and the salt concentration maybe high for this protein. I will try pH 9 buffer and remove salts.
Dear Mr Haque,
this is a problem I also encountered several years ago. I found some good hints in this publication:
Fink, A. L. (1998). Protein aggregation: folding aggregates, inclusion bodies and amyloid. Folding and design, 3(1), R9-R23.
I tested the addition of arginine and it improved solubility of my protein a bit. You can also test, whether low percentages of ethanol might have an influence as it can be also a stabilizing agent for e.g. albumin (1 - 3 % (v/v)).
kindly
JR
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